1. Technical Field
This invention relates to the field of genetically engineered, redirected T cells and to the field of cellular immunotherapy of malignancies such as Non-Hodgkin""s lymphoma and lymphocytic leukemia.
2. Description of Related Art
Over 30,000 new cases of Non-Hodgkin""s lymphoma are diagnosed each year in the United States alone. (Shipp et al., Cancer: Principles and Practice of Oncology, Lippincott-Raven Publishers, Philadelphia, 1997, p2165). While current therapies have produced significant complete response rates, a large percentage of patients remain at significant risk for disease relapse (Glass et al., Cancer 80:2311, 1997). Immune-based strategies for targeting minimal residual disease are under development and may provide additional modalities for consolidating standard chemotherapy and radiotherapy regimens. The approach of treating lymphoma with adoptive T cell therapy is predicated on the assumptions that tumor-reactive T cells can be isolated from individuals with lymphoma and expanded in vitro, and that infusion of the expanded effector population into the patient will mediate an antitumor effect without significant toxicity. Adoptively transferred donor-derived Epstein-Barr virus (EBV)-specific T cells can eliminate transformed B cells as demonstrated in the setting of post-transplant EBV-associated lymphoproliferative disease (Heslop et al., Immunol. Rev. 157:217, 1997). The clinical application of cellular immunotherapy for lymphoma using autologous T cells is currently limited by the paucity of molecularly-defined lymphoma target antigens for T cell recognition and the challenges of reliably isolating and expanding tumor-antigen specific T cell responses from cancer patients.
In order to overcome these obstacles, we and others are evaluating chimeric antigen receptor constructs consisting of a monoclonal antibody single chain Fv (scFv) linked to the intracellular signaling domain of CD3 zeta or Fcxcex3RIII for the purpose of re-directing T cell specificity. This strategy allows for the targeting of tumor cells based on the binding of the scFv portion of the receptor to monoclonal antibody-defined cell-surface epitopes. The capacity of these receptors when expressed in T cells to trigger cytokine production and cytolysis in vitro is now well established in both murine and human T cells. See Gross et al., FASEB J. 6:3370, 1992; Eshhar et al., PNAS USA, 90:720, 1993; Stancovski et al., J. Immunol., 151:6577, 1993; Moritz et al., PNAS USA 91:4318, 1994; Hwu et al., Cancer Res., 55:3369, 1995; Weitjens et al., J. Immunol. 157:836, 1996. Animal model systems demonstrate the capacity of murine T cell transfectants to eradicate tumor in vivo, suggesting that these gene-modified cells retain appropriate homing and recycling mechanisms (Hekele et al., Int. J. Cancer 68:232, 1996). This system is not dependent on pre-existing antitumor immunity since the generation of tumor-reactive T cells for therapy can be accomplished by the genetic modification of polyclonal T cells present in peripheral blood. Moreover, target epitope recognition by scFv is not HLA-restricted, thereby permitting the use of receptor constructs in populations of lymphoma patients irrespective of HLA differences.
A critical aspect of this chimeric receptor strategy is the selection of target epitopes that are specifically or selectively expressed on tumor, are present on all tumor cells, and are membrane epitopes not prone to shed or modulate from the cell surface. Nearly 80% of Non-Hodgkin""s lymphoma are B cell in origin and are defined in part by the cell surface expression of the CD20 molecule. This 33-37 KD protein is uniformly expressed on normal B cells and malignant B cells at a density greater than 12,000 molecules per cell (Vervoordeldonk et al., Cancer 73:1006, 1994). CD20 does not modulate or shed from the cell surface and has structural features consistent with that of an ion channel (Press et al., Blood 83:1390, 1994; Bubien et al., J. Cell Biol. 121:1121, 1993). The United States Food and Drug Administration (FDA) has approved a chimeric CD20-specific monoclonal antibody (rituximab) for lymphoma therapy. Initial clinical experience with CD20-targeted immunotherapy suggests that malignant B cells may have a limited capacity to down regulate CD20 expression. These attributes make CD20 an attractive target for genetically engineered, redirected T cells.
CD8+ cytolytic T cells (CTL) are immunologic effector cells that have the capacity to specifically recognize and directly lyse target cells (Henckart, Semin. Immunol. 9:85, 1997). Re-infusion of ex vivo expanded tumor-specific CD8+ CTL clones can mediate tumor eradication as demonstrated in animal model systems (Greenberg, Adv. Immunol. 49:281,1991). A growing number of genes encoding proteins expressed by human tumors that elicit T cell responses have been identified by expression cloning technologies. (Robbins et al., Current Opin. Immunol. 8:628, 1996; De Plaen et al., Methods 12:125, 1997). The feasibility of isolating T cells from cancer patients with specificity for these molecularly defined tumor antigens is currently being evaluated but remains a significant challenge to the clinical application of adoptive T cell therapy for malignant disease (Yee et al., J. Immunol. 157:4079, 1996).
Endowing T cells with tumor specificity by gene transfer of cDNA constructs encoding engineered antigen receptors is an alternate strategy for generating tumor-reactive CTL for therapy. (Weiss et al., Semin. Immunol. 3:313, 1991; Gross et al., supra; Hedrick et al., Int. Rev. Immunol. 10:279, 1993). These cell-surface chimeric molecules are distinguished by their ability to both bind antigen and transduce activation signals via immunoreceptor tyrosine-based activation motifs (ITAM""s) present in their cytoplasmic tails. Receptor constructs utilizing an antigen-binding moiety generated from single chain antibodies (scFv) afford the additional advantage of being xe2x80x9cuniversalxe2x80x9d in that they bind native antigen on the target cell surface in an HLA class I independent fashion. Several laboratories have reported on scFv constructs fused to sequences coding for the intracellular portion of the CD3 complex""s zeta chain (xcex6), the Fc receptor gamma chain, and sky tyrosine kinase (Eshhar et al., supra; Fitzer-Attas et al., J. Immunol. 160:145, 1998). Re-directed T cell effector mechanisms including tumor recognition and lysis by CTL have been documented in several murine and human antigen-scFv:xcex6 systems (Eshhar, Cancer Immunol. Immunother. 45:131, 1997; Altenschmidt et al., J. Mol. Med. 75:259, 1997; Brocker et al., Adv. Immunol. 68:257, 1998.
Clinical cellular immunotherapy trials have utilized gene-modified T cells for gene marking purposes, the expression of suicide genes permitting in vivo ablation of transfected cells, the expression of genes designed to protect T cells from HIV infection, and the expression of chimeric antigen receptors (Rosenberg et al., Human Gene Therapy 8:2301, 1997). A growing number of applications of T cell gene therapy for manipulating T cell survival, trafficking, and effector functions are under development for clinical application. To date, retroviral vectors remain the preeminent modality for gene transfer into primary human T cells. These vectors provide for relatively high transduction efficiencies and stable chromosomal integration but place constraints on the sequence and amount of cDNA which can be packaged and are difficult, time consuming, and expensive to produce as clinical grade material. A gene transfer system that provides a high degree of flexibility with respect to the configuration and sequence of cDNA constructs, that can be rapidly modified, and that is non-infectious and inexpensive to produce as a clinical reagent, may provide a viable alternative to retroviral systems.
Plasmid DNA represents a highly versatile platform for constructing expression cassettes that are active in mammalian cells. When combined with electroporation, a procedure by which DNA is introduced into cells through transient pores formed in the plasma membrane following exposure to brief electrical current, a simple and easily applied gene transfer system is created. Although transformed human lymphoid cell lines are amenable to stable transfection by electroporation of plasmid vectors, primary human T cells have been regarded to be resistant to this methodology for stable modification (Ebert et al., Gene Ther. 4: 296, 1997; Gallot et al., Blood 88:1098, 1996).
In one aspect, this invention provides genetically engineered T cells which express and bear on the cell surface membrane a CD20-specific chimeric T cell receptor having an intracellular signaling domain, a transmembrane domain and an extracellular domain. The extracellular domain comprises a CD20-specific receptor. Individual T cells of the invention may be CD4+/CD8xe2x88x92, CD4xe2x88x92/CD8+, CD4xe2x88x92/CD8xe2x88x92 or CD4+/CD8+. The T cells may be a mixed population of CD4+/CD8xe2x88x92 and CD4xe2x88x92/CD8+ cells or a population of a single clone. CD4+ T cells of the invention produce IL-2 when co-cultured in vitro with CD20+ lymphoma cells. CD8+ T cells of the invention lyse CD20+ human lymphoma target cells when co-cultured in vitro with the target cells. The invention includes the CD20-specific chimeric T cell receptors, DNA constructs encoding the receptors, and plasmid expression vectors containing the constructs in proper orientation for expression.
T cells of the invention are referred to in this specification as CD20-specific redirected T cells.
In another aspect, the invention is a method of treating a CD20+ malignancy in a mammal which comprises administering CD8+ CD20-specific redirected T cells to the mammal in a therapeutically effective amount. The CD8+ T cells are preferably administered with CD4+ CD20-specific redirected T cells. In another aspect, the invention is a method of treating a CD20+ malignancy in a mammal which comprises administering CD4+ CD20-specific redirected T cells and CD8+ cytotoxic lymphocytes which do not express the CD20-specific chimeric receptor of the invention, optionally in combination with CD8+ CD20-specific redirected T cells. The invention includes a method of purging CD20+ leukemic stem cells following autologous transplantation for leukemia by administering CD20-specific redirected T cells.
In another aspect, the invention is a method of abrogating any untoward B cell function in a mammal which comprises administering to the mammal CD20-specific redirected T cells in a therapeutically effective amount. These can include antibody mediated autoimmune disease (e.g., lupus or rheumatoid arthritis) as well as any unwanted specific immune response to a given antigen. For example, CD20-specific redirected T cells can be administered in a method of immunosuppression prior to administering a foreign substance such as a monoclonal antibody or DNA or virus or cell in the situation where any immune response would decrease the effectiveness of the foreign substance.
In a preferred embodiment, the CD20-specific redirected T cells express CD20-specific chimeric receptor scFvFc:xcex6 where scFv designates the VH and VL chains of a single chain monoclonal antibody to CD20, Fc represents at least part of a constant region of an IgG1, and xcex6 represents the intracellular signaling domain of the zeta chain of human CD3. The extracellular domain scFvFc and the intracellular domain xcex6 are linked by a transmembrane domain such as the transmembrane domain of CD4. In a specific preferred embodiment, the scFvFc:xcex6 is amino acids 21-633 of Seq. ID No. 2 encoded by DNA construct Seq. ID No. 1.
The invention includes a method of making and expanding the CD20-specific redirected T cells which comprises transfecting T cells with an expression vector containing a DNA construct encoding the CD20-specific chimeric receptor, then stimulating the cells with CD20+ cells, recombinant CD20, or an antibody to the receptor to cause the cells to proliferate.
In another aspect, this invention is a method of stably transfecting and re-directing T cells by electroporation using naked DNA. Most investigators have used viral vectors to carry heterologous genes into T cells. By using naked DNA, we can reduce significantly the time required to produce redirected T cells. xe2x80x9cNaked DNAxe2x80x9d means DNA encoding a chimeric T cell receptor (TCR) contained in a plasmid expression vector in proper orientation for expression. The electroporation method of this invention produces stable transfectants which express and carry on their surfaces the chimeric TCR (cTCR). xe2x80x9cChimeric TCRxe2x80x9d means a receptor which is expressed by T cells and which comprises intracellular signaling, transmembrane and extracellular domains, where the extracellular domain is capable of specifically binding in an MHC unrestricted manner an antigen which is not normally bound by a T cell receptor in that manner. Stimulation of the T cells by the antigen under proper conditions results in proliferation (expansion) of the cells and/or production of IL-2. The CD20-specific chimeric receptor of this invention is an example of a chimeric TCR. However, the method is applicable to transfection with chimeric TCRs which are specific for other target antigens, such as chimeric TCRs that are specific for HER2/Neu (Stancovski et al., supra) ERBB2 (Moritz et al., supra), folate binding protein (Hwu et al., supra), renal cell carcinoma (Weitjens et al., supra), and HIV-1 envelope glycoproteins gp120 and gp41 (Roberts et al., Blood 84:2878, 1994).
In a preferred embodiment of transfection method of the invention, the T cells are primary human T cells, such as human peripheral blood mononuclear cells (PBMC), which have previously been considered resistant to stable transfection by electroporation of plasmid vectors. Preferred conditions include the use of DNA depleted of endotoxin and electroporation within about 3 days following mitogenic stimulation of T cells. Following transfection, the transfectants are cloned and a clone demonstrating presence of a single integrated unrearranged plasmid and expression of the chimeric receptor is expanded ex vivo. The clone selected for expansion preferably is CD8+ and demonstrates the capacity to specifically recognize and lyse lymphoma target cells which express the target antigen. The clone is expanded by stimulation with IL-2 and preferably another stimulant which is specific for the cTCR such as, where the receptor includes the zeta chain of CD3, the monoclonal antibody OKT3.